Because of the complex organization and metabolic compartmentation in higher plants, many molecular tools have to be combined to successfully genetically manipulate a plant. At the present time, directed changes require a transformation system, a suitable gene, a promoter sequence capable of driving expression of the gene, effective targeting signals to direct the expression product to the correct destination in the cell, and often other regulatory elements.
There are many published systems for the transformation of plant cells. However, there remain drawbacks to each of the systems. For example, Agrobacterium tumefaciens gene transfer is widely used for creating transgenic plants, however, in some plants making a successful transformation utilizing the Agrobacterium system is difficult. Other systems include particle bombardment, viral vectors, protoplast transformation via polyethylene glycol or electroporation, microinjection of DNA into protoplast, and macroinjection of DNA. Of these, the most successful method to date is particle bombardment of embryogenic calli for developing embryos. Even this transformation system suffers from a lack of genomic targeting control for insertion of the foreign gene. In addition, bombardment results in multiple insertions of the foreign gene into the plant genome.
Any transformation system in plants, must deal with the problems of cosuppression which occurs when endogenous genes are down-regulated by expression of homologous sense transcripts. Expression of chimeric gene constructs have led to the down-regulation of genes in transgenic plants, however the effects of cosuppression are variable, and the underlying mechanisms remain unclear.
Site-directed manipulation of chromosomal genes has become the method of choice for determining gene function and bacterial, yeast, and mammalian cells. The primary methods used in site-directed gene manipulation rely on gene replacement via homologous recombination using an appropriately designed gene targeting vector. In plants, gene targeting has been limited by the frequency of homologous recombination. Even with improvements in transformation and selection methods, the frequency of gene targeting in plants is still 10−3-10−4 fold lower than random integration.
New procedures are being developed for mammalian gene therapy. One example is an approach using chimeric RNA/DNA oligonucleotides. In mammalian cells, chimeric oligonucleotides that contain both DNA/DNA and RNA/DNA duplex regions with homology to a target locus, are capable of specifically correcting mutations at a high frequency in both episomal and chromosomal target genes. Recently, mutations in liver alkaline phosphatase gene and factor IX gene have also been efficiently corrected in human hepatoma cells. However, to date, chimeric oligonucleotide-based gene targeting has not been reported in plant systems.
Herbicide resistant forms of plants are desirable for many breeding and crop production applications. Approaches to date have involved laborious methods including: finding a naturally existing form of resistance in a plant and introgressing the trait into desirable germplasm; mutagenesis of plants, seeds, and seedlings to generate novel mutant plants that confer resistance and introgressing the trait into the breeding population; finding a naturally existing form of a gene which confers resistance to a target herbicide and introducing the gene into the desired species by transformation; and, converting a wild type gene to a resistant form by mutagenesis. All of these approaches rely on either natural recovery of the trait or modification of the gene and subsequent introduction of the resistance gene into a plant.
A major disadvantage in each of these approaches is the time involved in terms of mutagenesis, recovery of the trait and the breeding necessary to introduce the trait into desired populations. Further, where transformation is involved, plants will have to be tested and selected that are not impacted by expression instability or by poor agronomic performance. Additionally, in many instances, optimum performance of a gene in a given species may only be achieved following resynthesis of the gene to maximize usage of preferred codons, or by the creation of modified forms of the gene.
Because of the present problems associated with the integration and expression of foreign genes in plant cells, effective strategies for modification, conversion or correction of gene sequences are needed.